ProteoChip: A highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein‐protein interaction studies

Abstract

We have developed a highly sensitive microarray protein chip, ProteoChip, coated with ProLinker, novel calixcrown derivatives with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrixes and makes high-throughput analysis of protein-protein interactions possible. The analysis of quartz crystal microbalance showed that both monoclonal antibody (mAb) and antigen (Ag) bound to the gold film of the sensor surface coated with ProLinker B and that it is useful for studies of Ab-Ag interactions. ProteoChip, aminated glass slide coated with ProLinker A, was also demonstrated to be useful for preparation of high-density array spots by using a microarrayer and for analysis of analyte Ags either by direct or sandwich methods of fluorescence immunoassay. The detection sensitivity of ProteoChip was as low as 1-10 femtogram/mL of analyte protein, useful for detection of tumor markers. ProteoChip was also useful for studies of direct protein-protein interactions as demonstrated by analysis of integrin-extracellular matrix protein interaction. These experimental results suggest that ProteoChip is a powerful tool for development of chip-based lead screening microarrays to monitor protein-protein interactions (i.e. drug target) as well as for biomarker assays which require high detection sensitivity.

Capabilities of Piezoelectric Immunosensors for Detecting Infections and for Early Clinical Diagnostics

“…In order to determine low concentrations of tumour markers, various techniques aimed at increasing detection sensitivity are used. One of the methods of immobilizing bioreceptor molecules is to use nanoparticles of various nature -magnetic (Chen et al 2007, Wei et al 2010 or gold (Tang et al 2006, Tang et al 2008, Uludağ and Tothill 2010, as well as calixarene (Lee et al 2003).…”

“…In order to increase the stability of the biolayer a method of immobilizing protein molecules (antigens or antibodies) is proposed using the bifunctional linkers and calixarenes "Prolinkers" (Lee et al 2003). It is shown that the activation of a solid surface by Prolinkers (Fig.…”

Trends and Perspectives in Immunosensors

“…Novel surfaces for Ab adsorption with improved orientation have recently been reported. Surfaces covered with calixarene derivatives have been demonstrated to capture Abs with a proper orientation [10,11]. In addition, Zn II metal complexes have been used for Ab adsorption [12,13].…”

Current perspectives in protein array technology

“…A recent a⁄nity-based method uses calixcrown-5 derivatives. 40 An aldehyde or thiol-activated calixcrown derivative is attached to the substrate surface. Proteins can then bind to the immobilized calixcrown via electrostatic interactions between the ionized amide groups on the protein and the crown moiety.…”